Antibiotic Susceptibility and Molecular Diversity of Bacillus anthracis Strains in Chad: Detection of a New Phylogenetic Subgroup

Abstract

We genotyped 15 Bacillus anthracis isolates from Chad, Africa, using multiple-locus variable-number tandem repeat analysis and three additional direct-repeat markers. We identified two unique genotypes that represent a novel genetic lineage in the A cluster. Chadian isolates were susceptible to 11 antibiotics and free of 94 antibiotic resistance genes.Bacillus anthracis, the etiological agent of anthrax, is a sporulating bacterium causing disease primarily in herbivores in many countries of Southern Europe, South America, Asia, and Africa (22). In Chad, anthrax is hyperendemic in cattle (22), and human cases of intestinal and cutaneous anthrax are reported each year (World Anthrax Data Site [http://www .vetmed.lsu.edu/whocc/]). However, studies of the genetic diversity and antibiotic susceptibilities of B. anthracis in this country have not been conducted. Here, we used a previously reported eight-marker multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) method (13) and three additional direct-repeat (DR) markers (15) to examine the genetic diversity of this pathogen in Chad. We also evaluated the Chadian B. anthracis isolates for antibiotic resistance by using physiological and genetic methods.Between 1996 and 2003, 174 blood samples were taken from the heart region or jugular vein of cattle carcasses in five southern prefectures in Chad. The samples were screened for B. anthracis by cultivation on tryptone soy agar plates containing 5% sheep blood. After incubation at 37°C for 48 h, cultures from 15 samples were identified as B. anthracis on the basis of colony morphology, absence of hemolysis, and susceptibility to B. anthracis-specific ␥ phage (2, 21). These cultures were sent to the Swiss Reference Centre (Bern, Switzerland) for further physiological and genetic testing.Template DNA for PCR was obtained by cell lysis followed by filtration (18). PCRs were performed using Taq DNA polymerase (Roche Diagnostics, Basel, Switzerland) and an annealing temperature of 55°C if not otherwise specified. We used PCR to screen the samples for the presence of B. anthracis chromosomal (Ba813) and plasmid (pXO1 and pXO2) sequences (see Table 1 for PCR targets and primer sequences). Eight VNTR markers, vrrA, vrrB 1 , vrrB 2 , vrrC 1 , vrrC 2 , CG3,

Bovine Bacillus anthracis in Cameroon

“…These six strains showed an additional base difference in the canSNP B.Br.004: the nucleotide just downstream of the canSNP is a thymine instead of a cytosine. We confirmed that this mutation is also present in all strains from Chad that were analyzed previously (12). One strain, JF3964, showed a completely different profile that has not yet been described (the characteristics of its canSNPs are A.Br.001, T; A.Br.002, A; A.Br.003, A; A.Br.004, T; A.Br.006, C; A.Br.007, T; A.Br.008, T; A.Br.009, A; B.Br.001, T; B.Br.002, C; B.Br.003, G; B.Br.004, T; and A/B.Br.001, A) and was assigned to a novel clade, designated clade D (Fig.…”

“…Strains JF3959, JF3060, JF3961, JF3963, JF3965, and JF3966 grouped in cluster A␤ (Fig. 1), together with B. anthracis strains from Chad, as previously determined (12). The close relationship of B. anthracis strains from Cameroon with those from Chad, with both belonging to cluster A␤, may be explained by the fact that these countries share a border in the northern part of Cameroon, where anthrax is hyperendemic and where most of the samples were collected.…”

“…However, our analysis showed that this cluster is identical to A␤, which appears to be a more appropriate designation since A␤ belongs to clade A (6, 22). (6,12,15). Strains from western and central Africa are boxed.…”

“…Thirteen canSNPs (A.Br.001, A.Br.002, A.Br.003, A.Br.004, A.Br.006, A.Br.007, A.Br.008, A.Br.009, B.Br.001, B.Br.002, B.Br.003, B.Br.004, and A/B. Br.001) and eight VNTR markers (VrrA, VrrB1, VrrB2, VrrC1, VrrC2, GC3, pXO1, and pXO2) for B. anthracis were amplified by PCR, using filtered lysates as template DNA (12), and sequenced. DNA sequencing was carried out with an ABI Prism 3100 genetic analyzer (Applied Biosystems, Rotkreuz, Switzerland) and a BigDye Terminator cycle sequencing kit (Applied Biosystems) according to the manufacturer's instructions.…”

Genetic Diversity in a Bacillus anthracis Historical Collection (1954 to 1988)

“…Discrepancies between the relative allele fragment sizes for MLVA markers, as determined with fragment analysis software, and the actual sizes determined by direct sequencing of PCR products and complete genome sequencing have been reported (3,18,19). These differences may occur if different instruments, software, fluorescent dyes, size standards, or other reagents are used.…”